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Realistic preclinical models of primary pancreatic cancer and metastasis are urgently needed to test the therapy response ex vivo and facilitate personalized patient treatment. However, the absence of tumor-specific microenvironment in currently used models, e.g., patient-derived cell lines and xenografts, only allows limited predictive insights. Organotypic slice cultures (OTSCs) comprise intact multicellular tissue, which can be rapidly used for the spatially resolved drug response testing. This protocol describes the generation and cultivation of viable tumor slices of pancreatic cancer and its metastasis. Briefly, tissue is casted in low melt agarose and stored in cold isotonic buffer. Next, tissue slices of 300 µm thickness are generated with a vibratome. After preparation, slices are cultured at an air-liquid interface using cell culture inserts and an appropriate cultivation medium. During cultivation, changes in cell differentiation and viability can be monitored. Additionally, this technique enables the application of treatment to viable human tumor tissue ex vivo and subsequent downstream analyses, such as transcriptome and proteome profiling. OTSCs provide a unique opportunity to test the individual treatment response ex vivo and identify individual transcriptomic and proteomic profiles associated with the respective response of distinct slices of a tumor. OTSCs can be further explored to identify therapeutic strategies to personalize treatment of primary pancreatic cancer and metastasis.

The use of in vitro systems to investigate the process of corneal wound healing offers the opportunity to reduce animal pain inflicted during in vivo experimentation. This study aimed to establish an easy-to-handle ex vivo organ culture model with porcine corneas for the evaluation and modulation of epithelial wound healing. Cultured free-floating cornea disks with a punch defect were observed by stereomicroscopic photo documentation. We analysed the effects of different cell culture media and investigated the impact of different wound sizes as well as the role of the limbus. Modulation of the wound healing process was carried out with the cytostatic agent Mitomycin C. The wound area calculation revealed that after three days over 90% of the lesion was healed. As analysed with TUNEL and lactate dehydrogenase assay, the culture conditions were cell protecting and preserved the viability of the corneal tissue. Wound healing rates differ dependent on the culture medium used. Mitomycin C hampered wound healing in a concentration-dependent manner. The porcine cornea ex vivo culture ideally mimics the in vivo situation and allows investigations of cellular behaviour in the course of wound healing. The effect of substances can be studied, as we have documented for a mitosis inhibitor. This model might aid in toxicological studies as well as in the evaluation of drug efficacy and could offer a platform for therapeutic approaches based on regenerative medicine.

BACKGROUND/AIMS: The use of skin-derived stem cells and stem cells of other origins in regenerative medicine requires knowledge of stem cell fate after transplantation. In order to achieve non-invasive long-term imaging and tracking of transplanted stem cells in preclinical studies, a non-toxic, efficient labeling technique that does not alter stem cell characteristics must be used. Our aim was to investigate a method for such a long-term cell-compatible cell tracer using nanoparticles. METHODS: Nanotechnology, in particular the use of quantum dots (QDs), offers great advantages for this crucial requirement. In this study, we used nanocrystals coated with a specific target peptide that enables delivery into the cytoplasm of cells, resulting in an intense and stable fluorescent labeling. We analyzed the influence of biocompatible CdSe/ZnS-QDs on epidermal stem cells (EpiSCs) isolated from adult human skin. Thereby we analyzed on QD loading, cell proliferation including QD transfer to descendent daughter cells as well as the influence on the differentiation potential of stem cells after QD labeling. RESULTS: FACS analysis revealed a dose-dependent QD incorporation into the cells. Thereby, a high initial concentration of nanocrystals resulted in a more stable long-term labeling. QD labeled cells showed normal viability and unchanged ability to proliferate. The spread of QDs during cell division was monitored by time lapse microscopy and two modes of QD distribution could be observed. Daughter cells either received an equal amount of QDs after cell division, which led to a homogenously faded fluorescence signal, or there was an uneven transmission of QDs, which led to unchanged labeling of one cell and a complete loss of the fluorescence signal of the other cell. The spontaneous differentiation potential remained unaffected after QD exposure, since skin-derived EpiSCs showed an unchanged protein and gene expression profile. CONCLUSION: In summary, we can conclude that QDs offer a successful, non-invasive and efficient labeling technique for EpiSCs, which makes their in vitro and in vivo use in skin regeneration and wound healing models traceable. Nevertheless, the uneven transmission of QDs should not be disregarded and the extent and frequency should be investigated in further studies.

Sweat glands play an important role in skin physiology and are an integral part of the natural skin barrier. In order to maintain functionality throughout life, sweat glands make use of several types of stem cells. This chapter focuses on the classification of different types of stem cells found in the sweat gland and their physiological roles. First, sweat gland formation during skin maturation is addressed in order to give an overview of sweat gland origin and formation in vivo. Then, different kinds of adult sweat gland stem cells are introduced and classified between different potency levels and corresponding physiological roles. Finally, the importance of these cell sources for future developments, including applications in wound healing and cosmetics research, is discussed.

The interaction of peripheral nerves with different cells of the skin is a relevant aspect of many physiological processes including nociception, temperature control, and wound healing. Here we describe a protocol for the setup of an indirect co-culture system of peripheral nerve cells and sweat gland-derived stem cells, which can be used to quantify neurite outgrowth.

The combination of an aging population and an increasing prevalence of diseases associated with impaired-wound healing, including obesity, peripheral vascular disease and diabetes, is likely to result in a dramatic increase in the incidence and prevalence of chronic skin wounds. Indeed, systemic reviews are now not only trying to establish both the prevalence and the often under-estimated socio-economic costs of chronic skin wounds, but most importantly are addressing the impact that chronic wounds have on quality of life. Given the clear need for novel approaches to the management of chronic skin ulceration, ideally developed and tested in the human system in a manner that can be rapidly translated into clinical practice, we examined the effects of multipotent primary human nestin + progenitor cells on human wound healing in an ex vivo model. Human sweat gland-derived nestin + cells demonstrated the capacity to significantly promote two key wound healing parameters, i.e., both reepithelialisation and angiogenesis in experimentally wounded, organ-cultured human skin. The current data further support the use of full-thickness human skin wound-healing models ex vivo to pre-clinically test wound healing-promoting candidate agents. Whilst larger studies are required to substantiate a firm “proof-of-concept,” our preliminary studies encourage further efforts to systemically determine the potential of cell-based regenerative medicine strategies in general, and the use of skin appendage-associated human nestin + cells in particular, as novel treatment strategies for chronic skin ulceration.

The interaction of peripheral nerves with different cells of the skin is a relevant aspect of many physiological processes including nociception, temperature control, and wound healing. Here we describe a protocol for the setup of an indirect co-culture system of peripheral nerve cells and sweat gland-derived stem cells, which can be used to quantify neurite outgrowth.

Sweat glands play an important role in skin physiology and are an integral part of the natural skin barrier. In order to maintain functionality throughout life, sweat glands make use of several types of stem cells. This chapter focuses on the classification of different types of stem cells found in the sweat gland and their physiological roles. First, sweat gland formation during skin maturation is addressed in order to give an overview of sweat gland origin and formation in vivo. Then, different kinds of adult sweat gland stem cells are introduced and classified between different potency levels and corresponding physiological roles. Finally, the importance of these cell sources for future developments, including applications in wound healing and cosmetics research, is discussed.

The combination of an aging population and an increasing prevalence of diseases associated with impaired-wound healing, including obesity, peripheral vascular disease and diabetes, is likely to result in a dramatic increase in the incidence and prevalence of chronic skin wounds. Indeed, systemic reviews are now not only trying to establish both the prevalence and the often under-estimated socio-economic costs of chronic skin wounds, but most importantly are addressing the impact that chronic wounds have on quality of life. Given the clear need for novel approaches to the management of chronic skin ulceration, ideally developed and tested in the human system in a manner that can be rapidly translated into clinical practice, we examined the effects of multipotent primary human nestin

The human skin fulfills important barrier, sensory, and immune functions—all of which contribute significantly to health and organism integrity. Widespread skin damage requires immediate treatment and coverage because massive skin loss fosters the invasion of pathogens, causes critical fluid loss, and may ultimately lead to death. Since the skin is a highly immunocompetent organ, autologous transplants are the only viable approach to permanently close a widespread skin wound. Despite the development of tissue-saving autologous transplantation techniques such as mesh and Meek grafts, treatment options for extensive skin damage remain severely limited. Yet, the skin is also a rich source of stem and progenitor cells. These cells promote wound healing under physiological conditions and are potential sources for tissue engineering approaches aiming to augment transplantable tissue by generating cultured epidermal autografts (CEAs). Here, we review autologous tissue engineering strategies as well as transplantation products based on skin-derived stem cells. We further provide an overview of clinical trial activities in the field and discuss relevant translational and clinical challenges associated with the use of these products.

Mammalian and fish skin share protective activities against environments that are rich in infectious agents. Fish epidermis is endowed with an extrinsic barrier consisting of a mucus layer and antimicrobial peptides (AMPs). These operate together as a protective chemical shield. As these AMPs are evolutionarily well preserved and also found in higher vertebrate skin (including human epidermis), fish skin offers a unique opportunity to study the origins of innate antimicrobial defense systems. Furthermore, the broad spectrum of fish mucus antimicrobial activities renders piscine AMPs interesting to investigative dermatology, as these may become exploitable for various indications in clinical dermatology. Therefore, this article aims at casting light on fish mucus, the evolutionary relationship between human and fish AMPs, and the latter's antibacterial, antifungal, and even antiviral activities. Moreover, we develop dermatological lessons from, and sketch potential future clinical applications of, fish mucus and piscine AMPs.

Increasing evidence suggests that vascular resident endothelial progenitor cells (VR-EPCs) are present in several organs, playing an important role in postnatal neovascularization. Here, we isolated and characterized VR-EPCs from cardiac tissue in vitro, evaluating their regenerative potential in vivo. VR-EPCs showed to be highly clonogenic and expressed several stem and differentiation markers. Under endothelial differentiation conditions, cells form capillary-like structures, in contrast to osteogenic or adipogenic differentiation conditions where no functional changes were observed. After seeding in scaffolds, cells were distributed homogeneously and directly attached to the scaffold. Then, cell seeded scaffolds were used to induce dermal regeneration in a nude mice full skin defect model. The presence of VR-EPCs enhanced dermal vascularization. Histological assays showed increased vessel number (p < 0.05) and cellularization (p < 0.05) in VR-EPCs group. In order to explore possible mechanisms of vascular regeneration, in vitro experiments were performed. Results showed that pro-angiogenic environments increased the migration capacity (p < 0.001) and ability to form capillary-like structures (p < 0.05) of VR-EPC. In addition, VR-EPCs secreted several pro-angiogenic molecules including VEGF and PDGF. These results indicate that a highly clonogenic population of VR-EPCs might be established in vitro, representing a new source for therapeutic vascularization in tissue engineering and regeneration.

We report the isolation, cultivation, and cryopreservation of cells from adipopancreatic tissue of adult Atlantic herring Clupea harengus. The cell population was cultured for >1 yr and had a mean doubling time of 2 wk. Immunocytochemical analyses revealed that >50% of cells contained glial fibrillary acidic protein and pan-cytokeratins. Vigilin, a well-conserved protein related to mRNA transport, was found in only 11% of cells. Alpha smooth-muscle actin and stage-specific embryonic antigen 1 were even less abundant (

Background/Aims: Cellular models are an interesting tool to study human heart diseases. To date, research groups mainly focus on mouse models, but important murine physiology is different from human characteristics. Recently, scientists found that the electrophysiology of fish cardiomyocytes largely resembles that of humans. So far, cardiomyocyte models were generated using differentiation medium, were stimulated electrically or, when contracting spontaneously, only did so over a short time period. We established an in vitro spontaneously, long-term beating heart model generated from rainbow trout, with the potential to be used as a new human heart model system because of its electrophysiology. Methods: Spontaneously contracting 3D cell layers from rainbow trout were generated in vitro and analyzed using PCR and immunochemistry. Further, electrophysiology was measured via intra – and extracellular recordings. Results: Contracting cardiomyogenic aggregates were generated without differentiation medium and were beating autonomously for more than one month. Electrophysiological measurements exhibit that the action potential properties of fish cardiomyocytes in part resemble the characteristics of human cardiomyocytes. The sensitivity of the beating cell aggregates to drugs could also be confirmed. Conclusion: Spontaneously contracting cardiomyogenic cell aggregates from rainbow trout generated in vitro are suitable for human heart research and pharmacology.

Cellular replacement therapies in vascular and urogenital organ disorders require an abundant source of smooth muscle cells. A promising approach would be the directed myogenic differentiation (characterized by the expression of alpha-smooth muscle actin (alpha-SMA)) into a sufficient amount of smooth muscle cells through easily obtainable adult stem cells, for example from the sweat gland.We present novel multipotent adult stem cell populations derived from glandular tissues like pancreas, salivary gland and sweat gland and assess their myogenic potential. Their possible application in cell replacement therapies is discussed, with regard to numerous scaffold-based approaches in the course of the last decade.Multipotent glandular stem cells can be manipulated by different means to express a predominant smooth muscle-like phenotype. Possible promising applications of myogenic differentiated stem cells were evaluated, since several studies revealed the beneficial effect of somatic stem cells in replacement therapies for blood vessels, bladder reconstructions, etc.Glandular stem cells, especially sweat-gland-derived cells, provide an easily accessible and efficient source for autologous smooth muscle tissue, which might be used to replace vascular tissue in case of organ failure or disorder.

Fish skin is a multi-purpose tissue that serves numerous vital functions including chemical and physical protection, sensory activity, behavioural purposes or hormone metabolism. Further, it is an important first-line defense system against pathogens, as fish are continuously exposed to multiple microbial challenges in their aquatic habitat. Fish skin excels in highly developed antimicrobial features, many of which have been preserved throughout evolution, and infection defense principles employed by piscine skin are still operative in human skin. This review argues that it is both rewarding and important for investigative dermatologists to revive their interest in fish skin biology, as it provides insights into numerous fundamental issues that are of major relevance to mammalian skin. The basic molecular insights provided by zebrafish in vivo-genomics for genetic, regeneration and melanoma research, the complex antimicrobial defense systems of fish skin and the molecular controls of melanocyte stem cells are just some of the fascinating examples that illustrate the multiple potential uses of fish skin models in investigative dermatology. We synthesize the essentials of fish skin biology and highlight selected aspects that are of particular comparative interest to basic and clinically applied human skin research.

Human skin-derived Nestin+ cells serve as a convenient source for autologous, adult, pluripotent progenitor cells that offer new therapeutic possibilities in cell-based regenerative medicine. However, the isolation of human Nestin+ cells has tended to be of very low efficiency and to produce highly variable cell yields. Here we report a standardised protocol that facilitates the isolation and enrichment of Nestin+ progenitor cells from enzymatically digested adult human scalp dermis. The use of distinct media like Dulbecco's modified Eagle medium supplemented with foetal bovine serum or, alternatively, serum-free, supplemented neural stem cell medium greatly affected cell morphology, proliferation and differentiation (e.g. towards a neural versus mesenchymal phenotype). Finally, Nestin+ cells were isolated from a heterogeneous dermis-derived progenitor cell population, which proliferates within clones or floating microspheres under defined serum-free culture conditions. Supplementation of the medium with epidermal growth factor and basic fibroblast growth factor as well as coating with fibronectin allowed the highest enrichment level of Nestin+ progenitors and differentiation towards neural fate. These methodological advances should greatly facilitate the isolation, culture and targeted differentiation of primary, adult human scalp skin dermis-derived Nestin+ cells.

Clinical success in tissue regeneration requires improvements in vascularization capacity of scaffolds. Several efforts have been made in this field including cellular and acellular technologies. In this work we combined the use of stem cells derived from pancreas or submandibular glands expressing green fluorescent protein (GFP(+)) with a commercially available scaffold for dermal regeneration. Cells were isolated, characterized and seeded in a scaffold for dermal regeneration. Scaffolds containing cells were used to induce dermal regeneration in a full skin defect model. After 3 weeks of in vivo regeneration, tissues were harvested and vascularization was analyzed. Results showed that gland-derived stem cells displayed stem cell features and presented multipotential differentiation capacity because they were able to differentiate in cell types representing the 3 different germ layers. After seeding, cells were homogeneously distributed and formed focal adhesions with the scaffold. Metabolic assays showed that cells can be cultured for at least 3 weeks in the scaffold. In vivo, the presence of pancreatic or submandibular stem cells significantly enhanced the vascularization compared to empty scaffolds. Presence of gland-derived stem cells in the regenerating tissue was confirmed by the detection of GFP expression in the wound area. In order to explore the possible mechanisms behind the improvement in vascular regeneration, in vitro experiments were performed, showing that gland-derived stem cells could contribute by angiogenic and vasculogenic mechanisms to this process. Our results suggest that the combined use of stem cells derived from glands and scaffold for dermal regeneration could be a rational alternative to improve vascularization in scaffold-mediated dermal regeneration.

Summary Background We have recently shown that the expression of nestin, a progenitor/stem cell marker protein, is localized in different mesenchymal compartments in human skin including the sweat gland stroma. Objectives As other exocrine glands are recognized sources of multipotent stem cell populations with potential for multilineage differentiation, it was our aim to isolate, expand and characterize glandular stem cells from human sweat glands. Methods Isolation of human sweat glands was based on mechanical and enzymatic digestion of axillary skin. Cultivation was performed on collagen-coated cell culture dishes and the resulting cell population was investigated at the protein and mRNA level. Results Outgrowing cells of isolated sweat glands showed a high-proliferation activity and were characterized by nestin expression in more than 80% of the cells. These sweat gland stem cells could be maintained in culture for long periods of time and showed spontaneous differentiation into cells representative of the different germ layers. Conclusions This pilot study provides the first, simple protocol for the isolation of adult human nestin-positive stem cells from the sweat gland mesenchyme, which promises to provide an easily accessible and abundantly available, autologous source of multipotent stem cells for cell-based regenerative medicine applications.

Glandular stem cells (GSCs) can be obtained from exocrine glands such as pancreas or salivary glands using well-established cell culturing methods. The resulting cell populations are characterized by a high proliferative capacity and an unusually high plasticity. Cells from pancreas have been demonstrated to differentiate into a multitude of cell types and even into oocyte-like cells. It has been found that the preparation method for GSCs can be applied to many vertebrates, including fishes and birds. Since the cells are excellently cryopreservable, this finding has been utilized to establish a new stem cell bank for preserving living cells of rare and wild animals. Apart from these advances, this mini-review also points out that GSCs from pancreas must not be confused with beta-cell progenitors but constitute a distinct cell type.

Summary In the application of adult stem cells in regenerative medicine, it is indispensable to control stem cell behaviour in vitro . Since stem cells spontaneously differentiate into several cell types, it is mandatory to identify methods to enrich the desired cell types and concurrently block other differentiation pathways. More precisely, generation of a defined cell population is a key prerequisite for a therapeutic application of stem cells. Here we have demonstrated that it is possible to influence the differentiation of human pancreatic stem cells (hPSCs). During activation of mesodermal differentiation, the cytoskeletal protein alpha-smooth muscle actin ( α -SMA) seems to play an important role in different cell systems and can usually be detected in hPSCs during in vitro cultivation. We cultured stem cells under different conditions and analyzed the impact on α -SMA expression. On the one hand, supplements like retinoic acid (RA) and dimethyl sulfoxide (DMSO) were added to the cultivation medium; on the other hand, different media with or without the addition of fetal calf serum (FCS) were used. Expression of α -SMA was determined by immunocytochemistry, Western blot analysis and quantitative RT-PCR. After the treatment of hPSCs with RA, a strong induction of α -SMA protein expression was observed when 2 mM RA was added to the medium. DMSO in turn induced a marked reduction in α -SMA-positive cells. This could also be observed using a keratinocyte serum-free medium (KSFM). Furthermore, the general addition of FCS to the medium had a blocking effect on α -SMA expression and decreased the number of α -SMA-positive cells to a minimum. The controlled modulation of hPSCs by soluble factors is a first success on the way to a promising application for transplantation medicine and cell therapy of degenerative diseases.

We have recently reported that the in vitro differentiation of human glandular stem cells into cardiac-like cells can be enhanced by co-culture with small myocardial biopsies. These results suggest that implantation of such cells directly into infarcted myocardium may facilitate the regeneration of the heart. As a preliminary to testing this approach in a goat model., pilot in vitro tests for these experiments have been performed and are presented here. Stem cells, isolated from the glandula submandibularis of Boer goats (SuSCs), have been co-cultured either directly or indirectly with heart biopsies from various species (Boer goat, rattus norwegicus, human) or heart conditioned medium for 48 h. We found a substantial increase in the number of cells expressing heart-specific marker proteins (Troponin I, Troponin T, sarcomeric myosin) regardless of the source organism of the heart biopsy. The proliferation of SuSCs also increased significantly under co-culture conditions. To benefit from these results in vivo, the stem cells must be delivered to the infarcted region in the heart and held securely in place over lengthy periods of time. Therefore, we repeated the co-culture experiments with SuSCs grown on biodegradable Vicryl (R)-meshes. The cells demonstrated good proliferation on the meshes and likewise, the expression of heart-specific marker proteins could be enhanced through co-culture with heart biopsies.

In vitro cultures of native fish cell lines are of great importance, both for basic research and applied science. In particular, there is strong demand for long-term growable cell lines from breeding fish, like sturgeon. Here, we describe the culture of cells from Siberian sturgeon (Acipenser baerii) head kidney. The cells have so far been cultured over a period of 12 months (24 passages). Cytochemical and immunocytochemical examination suggests that, in vitro, the cells exhibit markers that are indicative for different cell types. In particular, fat storing cells (adipocytes) were observed, and the expression of cytokeratins and glial fibrilar acidic protein (GFAP) can be concluded on the basis of immuncytochemical analysis. The observation of different morphologies additionally underlines the heterogeneity of the cell population and matches the typical behaviour of in vitro cultures of stem/progenitor cells. Different applications can be imagined.

The discovery of epithelial stem cells (eSCs) in the bulge region of the outer root sheath of hair follicles in mice and man has encouraged research into utilizing the hair follicle as a therapeutic source of stem cells (SCs) for regenerative medicine, and has called attention to the hair follicle as a highly instructive model system for SC biology. Under physiological circumstances, bulge eSCs serve as cell pool for the cyclic regeneration of the anagen hair bulb, while they can also regenerate the sebaceous gland and the epidermis after injury. More recently, melanocyte SCs, nestin+, mesenchymal and additional, as yet ill-defined "stem cell" populations, have also been identified in or immediately adjacent to the hair follicle epithelium, including in the specialized hair follicle mesenchyme (connective tissue sheath), which is crucial to wound healing. Thus the hair follicle and its adjacent tissue environment contain unipotent, multipotent, and possibly even pluripotent SC populations of different developmental origin. It provides an ideal model system for the study of central issues in SC biology such as plasticity and SC niches, and for the identification of reliable, specific SC markers, which distinguish them from their immediate progeny (e.g. transient amplifying cells). The current review attempts to provide some guidance in this growing maze of hair follicle-associated SCs and their progeny, critically reviews potential or claimed hair follicle SC markers, highlights related differences between murine and human hair follicles, and defines major unanswered questions in this rapidly advancing field.

Purpose Aqueous tear deficiency due to lacrimal gland insufficiency is one of the major causes of dry eye disease. In severe cases, such as Sjogren's syndrome, Stevens-Johnson syndrome, or ocular cicatricial pemphigoid, therapy with artificial tears is often insufficient to relieve severe discomfort, prevent progressive ocular surface disease, or enable visual rehabilitation by corneal transplantation. Cell or organ generation from stem cells, resulting in tear-like secretion, presents an option as a suitable alternative treatment. To obtain deeper insights into lacrimal gland stem cells we analyzed murine lacrimal glands for markers of pluripotency, self-renewal, and differentiation. Methods A special, patented technique with mechanical and enzymatic digestion was used to generate high numbers of cells in vitro from murine lacrimal glands. These presumptive "murine lacrimal gland stem cells" ("mLGSCs") can be propagated as monolayer cultures over multiple passages. By means of RT-PCR, Western blot, and immunohistochemistry, markers of pluripotency and differentiation were demonstrated. Hanging drop culture was used to build organoid bodies from mLGSCs to investigate their spontaneous differentiation in three-dimensional culture with histology, immunohistochemistry, and transmission electron microscopy methods. Results Isolated mLGSCs were cultured over more than 65 passages. Murine lacrimal gland stem cells expressed markers of pluripotency such as Nanog, Sox2, Kruppel-like factor 4 (Klf4), as well as early-lineage markers of all three germ layers. Three-dimensional culture of these cells revealed their ability to differentiate into various cell types. Conclusions Our results suggest that mLGSCs were isolated and cultured successfully. These cells have the ability to differentiate into all three germ layers. The results provide further insights into lacrimal gland stem cell physiology for engineering of a lacrimal gland construct to treat severe cases of tear deficiency in the future.

Cells isolated from pancreas have a remarkable potential for self-renewal and multilineage differentiation. We here present a comprehensive characterisation of stem/progenitor cells derived from exocrine parts of the adult rat pancreas. Using purified cells from either single colonies or even single-cell clones, we specifically demonstrate: (i) the cells contain the typical stem/progenitor cell markers alkaline phophatase, SSEA-1, Oct-4, CD9, Nestin, Pax6, CD44, a-Fetoprotein and Brachyury, demonstrated by immunocytochemistry and RT-PCR; (ii) the cells have the potential to differentiate into lineages of all three germ layers in vitro; (iii) a clonal analysis revealed that even cell lines derived from a single cell have stem/progenitor cell properties such as self-renewal and spontaneous differentiation into various cell lineages; (iv) the cells have the propensity to form three-dimensional, teratoma-like structures in vitro, which contain cells of different lineages; and (v) external stimuli can activate the generation of certain cell types. For instance, cells treated with retinoic acid show an increased expression of alpha-smooth muscle actin. These results suggest that exocrine glands, such as pancreas may be a potential source of adult stem/progenitor cells, suitable for cell therapy of degenerative diseases.

Pluripotent embryonic stem (ES) cells cultivated as cellular aggregates, so called embryoid bodies (EBs), differentiate spontaneously into different cell types of all three germ layers in vitro resembling processes of cellular differentiation during embryonic development. Regarding chondrogenic differentiation, murine ES cells differentiate into progenitor cells, which form pre-cartilaginous condensations in the EB-outgrowths and express marker molecules characteristic for mesenchymal cell types such as Sox5 and Sox6. Later, mature chondrocytes appear which express collagen type II, and the collagen fibers show a typical morphology as demonstrated by electron-microscopical analysis. These mature chondrogenic cells are organized in cartilage nodules and produce large amounts of extracellular proteoglycans as revealed by staining with cupromeronic blue. Finally, cells organized in nodules express collagen type X, indicating the hypertrophic stage. In conclusion, differentiation of murine ES cells into chondrocytes proceeds from the undifferentiated stem cell via progenitor cells up to mature chondrogenic cells, which then undergo hypertrophy. Furthermore, because the ES-cell-derived chondrocytes did not express elastin, a marker for elastic cartilage tissue, we suggest the cartilage nodules to resemble hyaline cartilage tissue.

Adult stem cells are undifferentiated cells found within fully developed tissues or organs of an adult individuum. Until recently, these cells have been considered to bear less self-renewal ability and differentiation potency compared to embryonic stem cells. In recent studies an undifferentiated cell type was found in primary cultures of isolated acini from exocrine pancreas termed pancreatic stellate cells. Here we show that pancreatic stellate-like cells have the capacity of extended self-renewal and are able to differentiate spontaneously into cell types of all three germ layers expressing markers for smooth muscle cells, neurons, glial cells, epithelial cells, chondrocytes and secretory cells (insulin, amylase). Differentiation and subsequent formation of three-dimensional cellular aggregates (organoid bodies) were induced by merely culturing pancreatic stellate-like cells in hanging drops. These cells were developed into stable, long-term, in vitro cultures of both primary undifferentiated cell lines as well as organoid cultures. Thus, evidence is given that cell lineages of endodermal, mesodermal, and ectodermal origin arise spontaneously from a single adult undifferentiated cell type. Based on the present findings it is assumed that pancreatic stellate-like cells are a new class of lineage uncommitted pluripotent adult stem cells with a remarkable self-renewal ability and differentiation potency. The data emphasize the versatility of adult stem cells and may lead to a reappraisal of their use for the treatment of inherited disorders or acquired degenerative diseases.

The biological relevance of vigilin a ubiquitous multi (KH)-domain protein is still barely understood. Investigations over the last years, however, provided evidence for a possible involvement of vigilin in the nucleo-cytoplasmic transport of tRNA and in the subsequent association of tRNA with ribosomes. We therefore investigated the potential association of vigilin with 80S ribosomes. Immunostaining, gel filtration, westernblot analysis of polyribosomes and high salt treatment of 80S ribosomes isolated from fresh human placenta were applied to analyze the possible association of vigilin with ribosomes. Overlay assays were performed to examine whether vigilin is capable of binding to ribosomal proteins. Immunostaining of HEp-2 cells, gel filtration of a cytoplasmic extract of HEp-2 cells and westernblot analysis of isolated 80S ribosomes clearly demonstrate that vigilin is bond to the ribosomal complex. Vigilin detaches from the ribosomal complex under the influence of high salt concentrations. We present data that radioactively labeled human vigilin interacts directly with a subset of ribosomal proteins from both subunits. We were able to narrow down the putative binding region to the C-terminal domain by using vigilin mutant constructs. Therefore our results provide strong evidence that vigilin is bond to the ribosomal complex and underline the hypothesis that vigilin might be involved in the link between tRNA-export and the channeled tRNA-cycle on ribosomes.

The-multi-KH domain protein vigilin has been identified by ex vivo experiments as both a tRNA- and/or mRNA-binding protein. We show here that in vitro under conditions previously shown to allow tRNA binding, recombinant vigilin also binds to selected mRNA species and ribosomal RNA. An in vivo link of vigilin to mRNA and rRNA was elucidated by several approaches. (i) Coexpression/costimulation of vigilin was found with many other proteins independently of whether their mRNA was translated on free or membrane-bound ribosomes. (ii) A close codistribution of vigilin with free ribosomes was seen in the cytoplasm while nucleoli were a major organelle of vigilin accumulation in the nucleus. (iii) Furthermore, free and membrane-bound ribosomes can be enriched for vigilin which suggests that this binding does not depend on the class of mRNA translated. Therefore, we suggest that vigilin does not distinguish between free or membrane-bound ribosomes but is generally necessary for the localization of mRNAs to actively translating ribosomes.

Vigilin, a K homology (KH) protein has been found in all eukaryotic species studied. It has a unique structure of 14-15 consecutively arranged KH domains which apparently mediate RNA-protein binding. Cloning and sequencing of the mouse vigilin cDNA confirmed that the amino acid sequences of vertebrate vigilins are highly conserved and contain conserved sequence motifs of nuclear import and export sequences. The human and murine vigilin mRNAs carry two alternatively spliced 5' exons. In the 5' leader region of one of the splice variants, variant 1A, we found an upstream open reading frame (uORF) highly conserved between mouse and human. Here we present for the first time evidence that a 13 amino acid long peptide encoded by this uORF is an inhibitor of vigilin expression operating on a posttranscriptional level. We propose that the two structurally different 5' leader sequences of the human vigilin mRNA are involved in the regulation of vigilin biosynthesis.

A long term functional and reliable coupling between neural tissue and implanted microelectrodes is the key issue in acquiring neural electrophysiological signals or therapeutically excite neural tissue. The currently often used rigid micro-electrodes are thought to cause a severe foreign body reaction resulting in a thick glial scar and consequently a poor tissue-electrode coupling in the chronic phase. We hypothesize, that this adverse effect might be remedied by probes compliant to the soft brain tissue, i.e., replacing rigid electrodes by flexible ones. Unfortunately, this flexibility comes at the price of a low stiffness, which makes targeted low trauma implantation very challenging. In this study, we demonstrate an adaptable and simple method to implant extremely flexible microprobes even to deep areas of rat's brain. Implantation of flexible probes is achieved by rod supported stereotactic insertion fostered by a hydrogel (2% agarose in PBS) cushion on the exposed skull. We were thus able to implant very flexible micro-probes in 70 rats as deep as the rodent's subthalamic nucleus. This work describes in detail the procedures and steps needed for minimal invasive, but reliable implantation of flexible probes.

Introduction: Nestin-expressing cells isolated from different human tissues reveal self-renewal capacity and a multilineage differentiation potential. In particular, adult stem/progenitor cell populations from exocrine glands such as the pancreas, salivary gland and sweat gland are characterized by prominent nestin expression. Interestingly, human mammary gland histological examinations also demonstrated the existence of nestin-positive cells in the ductal compartments. Within the scope of our previous work we wonder whether an isolation of nestin-positive cell populations from human mammary gland biopsies is possible and what characteristics they have in vitro. Cell populations from both sexes were propagated and subjected to a comparison with other gland-derived cell populations. Methods: Human mammary tissue biopsies were mechanically and enzymatically treated, and the isolated acini structures were observed with time-lapse microscopy to track adherently outgrowing cells. The proliferation potential of the cell population was assessed by performing growth curves. On the gene and protein levels we investigated the expression of stem cell markers as well as markers indicating multilineage differentiation. Results: We succeeded in establishing proliferating cell populations from breast tissue biopsies of both sexes. Our results display several similarities to the glandular stem cell populations from other exocrine glands. Beside their proliferation capacity during in vitro culture, the obtained cell populations are characterized by their prominent nestin expression. The cells share surface proteins commonly expressed on adult stem cells. We demonstrated the expression of stem cell-related genes like Oct4, Sox2, KLF4 and Nanog, and confirmed multipotent differentiation capacity by detecting transcripts expressed in endodermal, mesodermal and ectodermal cell types. Conclusion: With this study we present an efficient procedure for isolation and propagation of nestin-positive stem cells obtained from male and female breast tissue, which is frequently available. The established multipotent cell populations could be easily expanded in vitro and thus hold promise for cell-based therapies and personalized medicine.

Human skin harbours multiple different stem cell populations. In contrast to the relatively well-characterized niches of epidermal and hair follicle stem cells, the localization and niches of stem cells in other human skin compartments are as yet insufficiently investigated. Previously, we had shown in a pilot study that human sweat gland stroma contains Nestin-positive stem cells. Isolated sweat gland stroma-derived stem cells (SGSCs) proliferated in vitro and expressed Nestin in 80% of the cells. In this study, we were able to determine the precise localization of Nestin-positive cells in both eccrine and apocrine sweat glands of human axillary skin. We established a reproducible isolation procedure and characterized the spontaneous, long-lasting multipotent differentiation capacity of SGSCs. Thereby, a pronounced ectodermal differentiation was observed. Moreover, the secretion of prominent cytokines demonstrated the immunological potential of SGSCs. The comparison to human adult epidermal stem cells (EpiSCs) and bone marrow stem cells (BMSCs) revealed differences in protein expression and differentiation capacity. Furthermore, we found a coexpression of the stem cell markers Nestin and Iα6 within SGSCs and human sweat gland stroma. In conclusion the initial results of the pilot study were confirmed, indicating that human sweat glands are a new source of unique stem cells with multilineage differentiation potential, high proliferation capacity and remarkable self renewal. With regard to the easy accessibility of skin tissue biopsies, an autologous application of SGSCs in clinical therapies appears promising.

Summary The expression of vigilin in the uterus of rats was investigated by immunoblotting and immunohistochemistry and compared to the ultrastructural features of the endometrial cells. Vigilin could not be detected in the uteri of ovariectomized rats. Administration of estrogen, alone or in combination with progesterone, significantly stimulated the expression of vigilin, mainly in the luminal and glandular epithelial cells. Ultrastructurally, these cells show the morphological features of an increased protein synthesis. Untreated mature rats demonstrate a cyclic pattern of vigilin expression with high levels during the estrogen dominated proestrus and early estrus stages and low levels a metestrus. The down-regulation of vigilin starts with the occurrence of apoptosis and autophagocytosis in the epithelium, but precedes the vanishing of the secretory granules. At diestrus the vigilin expression is intermediate and the vigilin staining of the epithelial cells is reduced. Huwever, the endometrial fibroblasts show a faint staining. Morphologically, these fibroblasts are characterized by large euchromatic nuclei and dilated cisternae of the rough endoplasmic reticulum. The results suggest that in the uterus of rats the expression of vigilin is stimulated by estrogen. Under the experimental conditions chosen no influence of progesterone on vigilin expression was detected.

It is still open to debate whether or not leptocephali actively ingest live food organisms. The uptake of food particles and their subsequent processing would depend on the presence of digestive enzymes capable of degrading proteins and other macromolecules. Thus, the determination of proteolytic activity and trypsin specific immunostaining have been used to identify tissue segments with the potential to degrade ingested proteins. Activity of trypsin was measured in intestinal structures along the entire fish and further localised to distinctive areas of the digestive tract by anti-eel trypsin immunohistochemistry. These results suggest that the digestive system in the larvae has the capacity to degrade macromolecules.

Vigilin is a member of the KH protein family and contains 14 tandemly arranged potential RNA-binding domains. Between KH domains 2 and 3 we have identified a nuclear localization sequence by cloning this sequence into the NH2-terminal region of phage T7 RNA polymerase as a reporter protein and by showing its transfer into the nucleus. Furthermore we provide experimental evidence that Vigilin is present both in the nucleus and in the cytoplasm in similar concentrations. These observations support the notion that Vigilin may shuttle between nucleus and cytoplasm presumably in contact with RNA molecules.

Background: Beside its anti-proliferative, anti-hypertensive and anti-inflammatory effects, heparin has shown the apoptotic effect in lymphoblasts. In the study, it is aimed to show the apoptotic effect of heparin in lymphoblasts with measuring intracellular calcium and using DNA analysis by flow cytometry, in vitro. Methods: Twenty-three newly diagnosed acute lymphoblastic leukemia patients were included in the study. We added 10 and 20 U/ml heparin into the seperated lymphoblast samples and determined the percentages of apoptosis and intracellular Ca++ levels at 0, 1 and 2 hours by flow cytometry, in vitro. Results: The apoptotic effect on the lymphoblasts were established in 10 and 20 U/ml heparin concentrations at 0, 1 and 2 hours (p=0.005). The apoptotic effect of heparin in lymphoblasts was higher at the first hour than those at 0 and 2 hours in 10 and 20 U/ml heparin concentrations (p=0.005). The highest apoptosis was determined in 20 U/ml heparin concentration at the first hour. Statistically significant increase in intracellular Ca++ levels were determined in 10 and 20 U/ml heparin concentrations at 1 and 2 hours (p=0.005). In 10 and 20 U/ml heparin concentrations, intracellular Ca++ levels were significantly higher at the first hour than 0 and 2 hours (p=0.005). The highest intracellular Ca++ concentration was determined in 20 U/ml heparin concentration at the first hour. Conclusion: Heparin induces apoptosis in lymphoblasts and intracellular Ca++ levels of the lymphoblasts synchronously increase with apoptosis. The increase of intracellular Ca++ level supports a concept that the mitochondria plays a role heparin-induced apoptosis in lymphoblasts.

Minimizing the foreign body response is seen as one critical research strategy for implants especially when designed for immune-privileged organs like the brain. The context of this work is to improve deep brain stimulating devices used in a consistently growing spectrum of psychomotor and psychiatric diseases mainly in form of stiff electrodes. Based on the compliance match hypothesis of biocompatibility we present another step forward using flexible implant materials covered with brain cell-mimicking layers. We covered two types of flexible polyimide films with glandular stem cells derived from pancreatic acini. Using real time-PCR and fluorescent immunocytochemistry we analyzed markers representing various cell types of all three germ layers and stemness. The results demonstrate an unchanged differentiation potential of the polyimide fixated cells as measured by mRNA and protein level. Additionally we developed a fibrinous hydrogel coating to protect them against she ar forces upon eventual implantation. By repeating previous analysis and additional metabolism tests for all stages we corroborate the validity of this improvement. Consequently we assume that a stem cell-containing cover may provide a native, fully and actively integrating brain-mimicking interface to the neuropil.

Vascularization is a key process in tissue engineering and regeneration and represents one of the most important issues in the field of regenerative medicine. Thus, several strategies to improve vascularization are currently under clinical evaluation. In the present study, stem cells derived from hu[for full text, please go to the a.m. URL], 49. Jahrestagung der Österreichischen Gesellschaft für Plastische, Ästhetische und Rekonstruktive Chirurgie (ÖGPÄRC), 42. Jahrestagung der Deutschen Gesellschaft der Plastischen, Rekonstruktiven und Ästhetischen Chirurgen (DGPRÄC), 16. Jahrestagung der Vereinigung der Deutschen Ästhetisch-Plastischen Chirurgen (VDÄPC)